A Supramolecular Strategy to Purify Protein Therapeutics

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I recall the poignant moment when my mother was diagnosed with a malignant lymphoma a few years ago. At that stressful hour of my life, a single ray of hope was the easily accessible advanced medical facilities available in this part of the world. Despite suffering from a number of side effects, my mother finally recovered from her illness over a course of six treatment cycles that lasted for about 18 weeks. During this period, one drug that particularly caught my attention was rituximab, which is a monoclonal antibody (mAb) targeting a specific B-cell surface antigen and sold with a brand name Rituxan®. I realized that the drug possessed not only a high potency but also a very high price.  My family was lucky since the treatment was affordable for us. However, someone who cannot afford this kind of costly treatment may be left in a sad predicament. One can imagine the infrastructure for cancer treatment that varies across different countries depending on their income levels. Especially, the insufficient medical capability in poorer countries makes proper diagnosis, care and treatment for cancer unaffordable. Realizing these present circumstances, it becomes a globally important issue to bring down the present day cost of cancer treatment. From a chemist’s point of view, bringing down the cost of preparation as well as purification of anti-cancer agents can be an affordable solution.

These days, the increasing number of expired patents for top selling biologics (See Table. 1) has facilitated the development of biosimilars,1 providing great potential to cut down the price by competition. Still, biologics and biosimilars are produced from living organisms with much higher complexity compared to small molecule drugs. This causes difficulties in their production and storage, and makes them costly. One of important and expensive steps in the production of biologics is their purification. Typically, affinity chromatography using protein A with high specificity towards immunoglobulin G (IgG), has been exploited as a golden-standard for the purification of monoclonal antibodies (biologics), although it possess some intrinsic problems including its high production cost, limited recyclability and leaching in presence of eluents. As alternatives, affinity-tag methods including His-Tag, C-Myc, GST have been used, but high cost and insufficient selectivity limits their usage. Thus, the biopharmaceutical industry still requires an efficient and versatile method for the purification of biologics.

Table 1. Top 10 selling drugs in the world in 20185

Rank

Drug

Trade name

Protein therapeutics

1

Adalimumab

Humira

2

Lenalidomide

Revlimid

×

3

Nivolumab

Opdivo

4

Pembrolizumab

Keytruda

5

Aflibercept

Eylea

6

Trastuzumab

Herceptin

7

Etanercept

Enbrel

8

Bevacizumab

Avastin

9

Rituximab

Rituxan

10

Rivaroxaban

Xarelto

×

As supramolecular chemists, we developed a useful tool for biotechnology using the unique host-guest chemistry of the pumpkin-shaped host molecule, cucurbit[7]uril (CB[7]) displaying ultrahigh binding affinity (Ka > 1013 M-1) toward specific guest molecules such as adamantylammonium (AdA), which is in par with that of the biotin-(strept)avidin pair. Interestingly, this high-affinity host-guest complex can be dissociated, when necessary, by treatment with competing guest molecules. We coined this controllable CB[7]-based non-covalent affinity binding pair as a supramolecular latching system 1,2. Taking advantage of this new chemical tool, we provided an efficient and versatile affinity purification of protein therapeutics using CB[7]-conjugated agarose beads (See Figure 1)3. Using this new purification strategy, we were successful in purifying a monoclonal antibody drug, Herceptin (widely used for treatment of breast cancer), as well as a much smaller therapeutic protein, Interferon alpha (used for treatment of leukemia) with high efficiency and high purity. In particular, by applying small and stable synthetic molecules, our research group succeeded in securing the manufacturability, sterilization, and recyclability of purified materials in a stable manner as well as in increasing the purity and productivity of purified protein therapeutics. In principle, by introducing the adamantly ammonium (AdA) functionality to any therapeutic proteins through genetic regulation and enzyme treatment one can purify them regardless of their size or type.

 Figure 1. Illustration of selective capture of AdA-labeled mAb using CB[7]-beads

Therefore, this technique can be applied to most recombinant therapeutic proteins, including antibodies or fusion proteins that effectively prevent or treat fatal diseases such as viral infections or cancer, and is highly efficient and reusable. Furthermore, it will be applicable to a wide variety of therapeutic proteins used in the development of vaccines or treatments, which will speed up their production. We hope our efforts can address some of the major global health issues.

To explore more details about our research, please read, “Purification of protein therapeutics via high-affinity supramolecular host–guest interactions” in Nature Biomedical Engineering (https://www.nature.com/articles/s41551-020-0589-7).

References:

  1. Moorkens, E., Vulto, A. G., Huys, I. An overview of patents on therapeutic monoclonal antibodies in Europe: are they a hurdle to biosimilar market entry? mAbs, 12, (2020), DOI: 10.1080/19420862.2020.1743517.
  2. Park, K. M., Murray, J. & Kim, K. Ultrastable artificial binding pairs as a supramolecular latching system: a next generation chemical tool for proteomics. Acc. Chem. Res. 50, 644–646 (2017)
  3. Kim, K. L. et al. Supramolecular latching system based on ultrastable synthetic binding pairs as versatile tools for protein imaging. Nat. Commun. 9, 1712 (2018).
  4. An, J. et al. Purification of protein therapeutics via high-affinity supramolecular host–guest interactions. Nat. Biomed. Eng. (2020) DOI:10.1038/s41551-020-0589-7.
  5. https://www.pharmalive.com/top-200-medicines-annual-report-2019-the-king-of-medicines/

Kyeng Min Park

Group leader , CSC, Institute for Basic Science (IBS)

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